Molecular Beacons for Tissue Engineering Applications
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A complete spatiotemporal profile of the mechanisms involved in the synthesis, processing, and transport of RNA will improve our understanding of cell function and behaviour under various biological cues, which could potentially advance the field of biomaterials and tissue engineering towards designing more functional biomaterial scaffolds Molecular beacons (MBs) have been extensively used in biomedical research and have the ability to provide spatiotemporal pattern of specific mRNA expression in live cells that can be visualized in real-time at the single cell level. The aim of this research was to develop a detection system that can be used to monitor gene expression in living cells associated with 3D scaffolds. The first stage of the research was dedicated to the design of molecular beacons targeting therapeutic genes of interest in cardiovascular tissue engineering. FAM-labelled DNA molecular beacons targeting human eNOS and IL-10 mRNAs were designed that were able to detect their nucleic acids targets with high specificity and sensitivity as well as access their intracellular mRNA targets in living cells emitting fluorescence signals that were stable for 2-3 h. The next stage was to develop a molecular beacon detection platform for monitoring changes in transcription gene expression levels in living cells embedded into a 3D collagen scaffolding system. For the first time the use of TAT-peptide conjugated MB to monitor mRNA in a 3D in vitro system was reported. It was shown that TAT-peptide linked molecular beacons can monitor GAPDH mRNA expression in 3D type I collagen scaffold of 1 mm thickness and that delivery can be completed with fast kinetics (~1h). Also, spatial distribution of cells in 3D can be visualized by optically sectioning the scaffolds. Although MB technology can be used to detect mRNA abundance in cells in 3D, it may be necessary to perform quantification with another complementary technique such as RT-PCR, unless next generation beacons can be stand alone. In the final stage of the research spatiotemporal efficacy of a dual-gene therapy model for cardiovascular tissue engineering based on a 3D collagen scaffold was assessed. It was found that IL-10 and eNOS MBs were able to detect changes in mRNA expression and demonstrated that the model supported both sustained and delayed release of functional genes, which was confirmed by RT-PCR results.